1. Technical Field
This disclosure relates generally to protease inhibitors and applications thereof, more specifically to peptide inhibitors of cysteine proteases, even more specifically to propenoyl hydrazides, methods of their use, and methods of their production.
2. Related Art
Protease inhibitors are important therapeutics in the treatment of a variety of disease conditions including viral infections such as HIV infection. Proteases are enzymes that cleave proteins or peptides and are classified into several groups. For example, cysteine proteases form a group of enzymes involved in numerous disease states, and inhibitors of these enzymes can be used therapeutically for the treatment of diseases involving cysteine proteases.
Cysteine Proteases.
Cysteine proteases employ a thiolate residue, which performs a nucleophilic attack on the amide bond of the peptide backbone to form a tetrahedral intermediate. The intermediate collapses to release the first product and the resulting acyl enzyme then undergoes hydrolysis. Based on their sequence homology cysteine proteases are divided into several clans and families. Clan CA and clan CD contain the majority of cysteine proteases. The majority of cysteine proteases, such as papain, calpains, cathepsins, and cruzain belong to the clan CA. According to the crystal structure of papain, clan CA proteases are unique for their catalytic triad formed by Cys, His, and Asn. The oxyanion hole is created by a preceding Gln residue. Clan CA enzymes are inhibited by E-64, a natural inhibitor of cysteine proteases, and cystatin. The substrate specificity of clan CA enzymes is primarily controlled by the S2 enzyme subsite. Clan CD enzymes are unique for their lack of inhibition by E-64 and their specificity for the P1 amino acid residue. Even though clan CD is the smallest of the clans, it contains some very important enzymes. Among them are caspases, legumains, gingipains, clostripain, and separase.
Caspases are a recently discovered family of cysteine endoproteases, which are highly selective for Asp at the P1 residue. As a result, this newly emerging family of proteases has been called caspases (cysteinyl aspartate—specific protease). All caspases contain the conserved pentapeptide active site motif Gln-Ala-Cys-X-Gly (QACXG)(SEQ. ID NO. 1), where X=Arg, Gln, Gly (R, Q, G), and are synthesized as inactive proenzymes. The only other mammalian protease with specificity for Asp is the lymphocyte serine protease, granzyme B. Many of the proteolytic cleavages that are observed during apoptosis and cytokine maturation are due to the action of various caspases. Indeed, many of the procaspases are activated by other caspases, which selectively cleave at P1 Asp residues in their recognition sites.
At present, there are 11 homologous members of the caspase family in humans. Some caspases are important mediators of inflammation, where they are involved in the production of inflammatory cytokines, and others are involved in apoptosis, where they participate in signaling and effector pathways. Group I caspases (caspases-1,-4, -5, -11, -12, -13, and -14) are primarily mediators of inflammation and are involved in proteolytic activation of proinflammatory cytokines. Caspase-1 is also involved in the Fas and TNFR apoptotic pathway. Group II caspases (caspases-2, -3, and -7) are late phase effectors of apoptosis and are involved in the cleavage of key structural and homeostatic proteins. Caspase-3, also known as CPP32 (cysteine protease protein 32-kDa), Yama or apopain, is believed to be one of the major effectors in apoptosis. This enzyme is a key executioner because it is responsible either partially or totally for proteolytic cleavage of key apoptotic proteins. It functions to decrease or destroy essential homeostatic pathways during the effector phase of apoptosis. Caspase-3 cleaves or activates nuclear enzymes, such as poly(ADP-ribose)polymerase (PARP), the 70 kDa subunit of the U1 small ribonucleoprotein, the catalytic subunit of DNA-dependent protein kinase, and protein kinase Cδ. Group III caspases (caspases-6, -8, -9, -10) are involved in the upstream early activation of effector caspases. Studies have shown that caspase-8 and -10 can cleave radiolabeled precursors for caspase-3. Caspase-6 is the only known caspase that cleaves the lamins, the major structural proteins in the nuclear envelope. Proteolysis of lamins is observed in cells undergoing apoptosis. Caspase-8 (MACH/FLICE), which can cleave all other known caspases, is suggested to lie in the pinnacle of the apoptotic cascade, at least when apoptosis is initiated by some stimuli such as Fas-L and TNF. Accordingly, the present disclosure encompasses compositions and methods of altering, inhibiting, or reducing the formation of enzymatic reaction products involving cysteine proteases. Inhibiting the formation of cysteine protease reaction products in vivo can provide therapeutic effects to patients suffering from unregulated or undesired protease activity.
Caspases have a specificity for at least four amino acids to the left of the cleavage site (P side). The S4 subsite is the single most important determinant of specificity among caspases after the P1 Asp. The optimal sequences of the caspases were obtained using a positional-scanning combinatorial substrate library (PS-CSL). The optimal recognition sequences for these enzymes are closely related to the sequences found in known macromolecular substrates. Group I caspases' optimal sequence is Trp-Glu-His-Asp (WEHD) (SEQ. ID NO. 2) with S4 favoring hydrophobic amino acids. Group II caspases' optimal sequence is Asp-Glu-X-Asp (DEXD) (SEQ. ID NO. 3) with a requirement for Asp in S4. Group III caspases' optimal sequence is N-Glu-X-Asp where N=Val or Leu, and X can be any amino acid ((V,L)EXD) (SEQ. ID NO. 4) with a preference for branched, aliphatic side chains in S4. The S3 subsite prefers glutamic acid (E) in most of the caspases, which could be explained by the salt link between Arg-341 (involved in stabilization of the P1 aspartic acid) and the glutamic acid in P3.
Legumains (EC.3.4.22.34, asparaginyl endopeptidase) form a another important family (C13) of clan CD proteases. They are related to caspases and other clan CD enzymes by a shared catalytic-site motif and a common scaffold within their catalytic domains. They were first identified in leguminous plants, and later in mammalian cells. In mammalian cells legumain has been linked to osteoclast formation and bone resorption, and the processing of bacterial antigens and the potential autoantigen, myelin basic protein, in the major histocompatability (MHC) class II system. In S. mansoni the legumain protease (SmAE, Sm32) is dissimilar to the other proteinases and may process gut-associated clan CA zymogens to their active forms, thus facilitating the digestion of ingested host serum proteins. S. mansoni legumain is not inhibited by the clan CA cysteine protease inhibitor E-64 or the diazomethane inhibitor Cbz-Phe-Ala-CHN2. Irreversible protease inhibitors would have great potential for the short-term therapeutic administration against parasitic infections. So far there has been no success in the determination of a crystal structure of the asparaginyl endopeptidase. Like all clan CD proteases, the substrate specificity of legumain is controlled by the interactions of the S1 subsite. Legumains selectively hydrolyze substrates with an asparaginyl residue in the P1 position. Legumain is less selective with respect to the P2 and P3 positions, but prefers Ala or Thr. The synthetic substrate Cbz-Ala-Ala-Asn-AMC was effectively cleaved by S. mansoni legumain and human legumain with KM's of 90 and 80 μM respectively.
Next to caspases and legumains, the family of separases has gained increasing attention among the clan CD enzymes. Separases play an important role during mitosis at the metaphase to anaphase transition. When the sister chromatids are aligned at the onset of the anaphase, separase initiates the segregation of the chromosomes by facilitating the breakdown of a cohesion complex between the individual sister chromatides.
Neural tissues, including brain, are known to possess a large variety of proteases, including at least two calcium-stimulated proteases termed calpains. Calpains are present in many tissues in addition to the brain. Calpain I is activated by micromolar concentrations of calcium while calpain II is activated by millimolar concentrations. In the brain, calpain II is the predominant form, but calpain I is found at synaptic endings and is thought to be the form involved in long term potentiation, synaptic plasticity, and cell death. Other Ca2+ activated cysteine proteases may exist, and the term “calpain” is used to refer to all Ca2+ activated cysteine proteases, including calpain I and calpain II. The terms “calpain I” and “calpain II” are used herein to refer to the micromolar and millimolar activated calpains, respectively, as described above. While calpains degrade a wide variety of protein substrates, cytoskeletal proteins seem to be particularly susceptible to attack. In some cases, the products of the proteolytic digestion of these proteins by calpain are distinctive and persistent over time. Since cytoskeletal proteins are major components of certain types of cells, this provides a simple method of detecting calpain activity in cells and tissues. Activation of calpains and/or accumulation of breakdown products of cytoskeletal elements have been observed in neural tissues of mammals exposed to a wide variety of neurodegenerative diseases and conditions. For example, these phenomena have been observed following ischemia in gerbils and rats, following stroke in humans, following administration of the toxins kainate, trimethyltin, or colchicine in rats, and in human Alzheimer's disease.
Cathepsin B is involved in muscular dystrophy, myocardial tissue damage, tumor metastasis, and bone resorption. In addition, a number of viral processing enzymes, which are essential for viral infection, are cysteine proteases. Inhibitors of cysteine proteases would have multiple therapeutic uses.
Other important cysteine proteases are the bacterial enzymes clostripain and gingipain. Gingipain causes tissue destruction during periodontal diseases.
Cysteine Protease Inhibitors.
To date, a structurally diverse variety of cysteine protease inhibitors have been identified. Palmer, (1995) J. Med. Chem., 38, 3193, discloses certain vinyl sulfones, which act as cysteine protease inhibitors for cathepsins B, L, S, O2 and cruzain. Other classes of compounds, such as aldehydes, niitriles, α-ketocarbonyl compounds, halomethyl ketones, diazomethyl ketones, (acyloxy)methyl ketones, ketomethylsulfonium salts and epoxy succinyl compounds have also been reported to inhibit cysteine proteases. See Palmer, id, and references cited therein. Many irreversible cysteine protease inhibitors have been described in the review by Powers, Asgian, Ekici, and James (2002) Chemical Reviews, 102, 4639. See Powers, id, and references cited therein. However, most of these known inhibitors are not considered suitable for use as therapeutic agents in animals, especially humans, because they suffer from various shortcomings. These shortcomings include lack of selectivity, cytotoxicity, poor solubility, and overly rapid plasma clearance.
Several types of Michael acceptor warheads have been employed as inhibitors for cysteine proteases. Among the most effective inhibitors are vinyl sulfones and α,β-unsaturated carbonyl derivatives against various cysteine proteases. Hanzlik, (1984) J. Med. Chem., 27, 711 has replaced the carbonyl group of a good substrate with a moiety, that would trap the enzymatic nucleophile (Ser-OH or Cys-OH) without altering the structural features required for enzyme recognition and binding. The fumarate derivative of the epoxy succinate E-64c, which is one of the first Michael acceptor inhibitors reported, extends the α,β-unsaturated carbonyl by an additional carbonyl for possible structural recognition and binding requirements within the enzyme active site. The fumarate derivative of E-64c (trans-HOOCCH═CH—CO-Leu-NH(CH2)2CH(CH3)2) inhibits cathepsin B (kapp=625 M−1s−1), cathepsin H (kapp=M−1s−1), and cathepsin L (kapp=2272 M−1s−1) irreversibly. Both the fumarate analog of E-64c and the epoxide parent compound do not inhibit clan CD proteases. Caspases, legumains, gingipains and clostripain are members of clan CD, while papain, cathepsins, and calpains are members of clan CA. Therefore, because of the aforementioned deficiencies in the art, there is a need for new compounds and methods for inhibiting proteases, in particular cysteine proteases.